Cochlear motion across the reticular lamina implies that it is not a stiff plate

Within the cochlea, the basilar membrane (BM) is coupled to the reticular lamina (RL) through three rows of piezo-like outer hair cells (OHCs) and supporting cells that endow mammals with sensitive hearing. Anatomical differences across OHC rows suggest differences in their motion. Using optical coherence tomography, we measured in vivo and postmortem displacements through the gerbil round-window membrane from approximately the 40–47 kHz best-frequency (BF) regions. Our high spatial resolution allowed measurements across the RL surface at the tops of the three rows of individual OHCs and their bottoms, and across the BM. RL motion varied radially; the third-row gain was more than 3 times greater than that of the first row near BF, whereas the OHC-bottom motions remained similar. This implies that the RL mosaic, comprised of OHC and phalangeal-process tops joined together by adhesion molecules, is much more flexible than the Deiters’ cells connected to the OHCs at their bottom surfaces. Postmortem, the measured points moved together approximately in phase. These imply that in vivo, the RL does not move as a stiff plate hinging around the pillar-cell heads near the first row as has been assumed, but that its mosaic-like structure may instead bend and/or stretch.

Supplementary Note 1. Cochlea sensitivity was monitored using distortion product otoacoustic emissions (DPOAEs) at approximately 20-min intervals during in vivo OCT vibrometry measurements. Following the injection of Fatal Plus, Postmortem DPOAEs were collected after the animal stopped breathing and had no heartbeat, at which point DPOAE amplitudes dropped to the noise floor level (data not shown). Supplementary Figure 1a shows DPOAEs responses from across animals (n=9) evoked by two simultaneous tones at frequencies f1 and f2 (f2/f1 = 1.2), with f2 swept from 2 to 63 kHz in octave steps below 30 kHz, and 2-kHz steps above 30 kHz. The two tones were presented with the f2 level 10 dB less than the f1 level, with the f2 level at 50 and 70 dB SPL in separate runs. A cochlea was considered to be healthy if the 2f1-f2 DP amplitudes from 50 dB SPL f1 tones were greater than a 10 dB SPL criterion. Figure 1. Cochlea sensitivity monitored using DPOAEs across animals (n=9). a: DPOAE amplitudes and noise floors measured at 2f1-f2, during in vivo OCT vibrometry. The symbols and solid lines represent the means and standard deviations of the DPOAE amplitudes, and the dashed lines represent the averaged noise floors, measured during the in vivo experiments (the durations of in vivo measurement times are shown in the legend). b: Average DP amplitudes near the best frequency (BF) frequency region, i.e., 38 kHz−48 kHz (gray box in a). The X axis is animal ID, sorted by animal numbers as in the main manuscript figures. Each animal's BF is labeled above its symbol. The black dashed horizontal line represents the 2f1-f2 DPOAE amplitude criterion of 10 dB SPL. All animals passed this criterion.

Supplementary Note 2.
Examples of reticular-lamina (RL) gain at three radial locations. The three radial locations (RL3, RL2, RL1) correspond to the apical surfaces of the three outer-hair-cell (OHC) rows. Shown are data from three animals including the one in the main manuscript (G637). The gains and phases from the living (dark colors) and postmortem (PM; faded colors) animals were normalized by the averaged BMAPJ gain from the corresponding animal, measured at a high level to remove the contribution of the BM traveling wave to RL motion. BM gains were measured at the junction of the arcuate zone (AZ) and pectinate zone ( . d-f: The phase responses corresponding to a-c. Note that the available stimulus levels vary across the structures due to different signal-to-noise ratios. Figure 9 presents the motions at three radial points along the BM (BMPZ, BMAPJ, and BMAZ), normalized by the high-level BMAPJ gain (in dB), in animal G637. Figure 9. Gerbil G637: a-c: In vivo and PM gains for BMPZ, BMAPJ, and BMAZ, respectively, normalized by the high-level BMAPJ gain. d-f: The phases corresponding to the magnitudes in a-c. The green dotted line shows the lowest-level BMAPJ data. The baseline high-level BMAPJ gain used for normalization the average of ten measurements made at 92 dB SPL.

Supplementary Note 5.
On gerbil G637 we measured motion of the pillar-cell head (PCH). In Supplementary Figure 10 we compare the in vivo BMAPJ, RL1, and PCH motions, normalized by the input sound pressure (left) and by the high-level BMAPJ gain (right. Near BF, BMAPJ, RL1, and PCH had non-linear compression (a, b). RL1 was more broadly tunned and had slightly higher gain than BMAPJ and PCH. At frequencies more than 1/2-oct below BF, the motion of PCH (magenta) was typically less than or equal to the motions of RL1 (blue) and BMAPJ (green). PCH had a broadly tunned dip of up to 10 dB near 30 kHz, while RL1 has a sharp dip near 25 kHz. For RL1, and PCH, the gain continued to grow above BF and was higher than at BF.
From low frequencies to BF in all three measurements, there was a phase change of about 3 cycles (c), consistent with a traveling wave. PCH phase was 0.15 cycles less than BMAPJ and RL1 at 20 kHz (d). RL1 phase led BMAPJ phase by 0.12 cycles near 25 kHz. The phase changed rapidly near BF for RL1 and PCH, but not as much for BMAPJ. This indicates a greater group delay for RL1 and PCH, than for BMAPJ, in this frequency region.
Supplementary Figure 10. a, b: In vivo gains of BMAPJ, RL1, and PCH normalized by sound pressure (left) and by averaged high-level BMAPJ gain (right) in Gerbil G637. The inset drawing shows the measurement locations for BMAPJ (green diamond) and RL1 (blue circle), and PCH (magenta tringle). c, d: The phases corresponding to the magnitudes in a, b. The blue dotted line shows the lowest-level RL1 measurement (46 dB SPL); this level was not measured on BMAPJ and PCH.